Cystinuria is an inherited defect of the renal tubules in which resorption of the amino acid cystine is impaired, urinary excretion is increased, and cystine calculi often form in the urinary tract. The diminished renal tubular resorption of cystine leads to an increase in cystine concentration in the urine. Since cystine is poorly soluble in acidic or neutral urine, the concentration of cystine in the urine can exceed its solubility, leading to supersaturation of urine with cystine and resulting in precipitation as cystine crystals or stones. Recurring cystine calculi can lead to urinary infection, obstruction, and possible loss of renal function. Treatment methods are aimed at reducing cystine excretion and increasing the solubility of cystine by means of (1) dietary restriction to limit cystine production and excretion, e.g., limiting the intake of sulfur-containing amino acids and reducing dietary sodium, (2) use of physical means to increase cystine solubility such as enhanced fluid intake or alkalinization of urine above pH 7.5, and (3) administration of drugs which contain thiol groups such as D-penicillamine, tiopronin, or captopril which bind cystine and convert it to a more soluble compound. Yet, despite the various treatment methods available, clinical reports indicate that formation of cystine stones continues to be a problem for cystinuric patients (Chow, G. K. and Streem, S. B. 1996. “Medical treatment of cystinuria: results of contemporary clinical practice,” J Urol 156:1576–1578; Pak, et al. 1986. “Management of cystine nephrolithiasis with alpha-mercaptopropionaoglycine,” J Urol 136:1003–1008).
Several qualitative and quantitative tests have been used to diagnose and treat cystinuria. One such test is a calorimetric method using sodium cyanide and sodium nitroprusside (Nakagawa, Y. and Coe, F. L. 1999. “A modified cyanide-nitroprusside method for quantifying urinary cystine concentration that corrects for creatinine interference,” Clin Chim Acta 28:57–68). Other preferred quantitative methods involves precolumn derivatization followed by reversed-phase high performance liquid chromatography (HPLC) or gas chromatography (Kaniowska, et al. 1998. “Urinary excretion measurement of cysteine and homocysteine in the form of their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection,” J Chromatog 798:27–35; Pastore, et al. 1998. “Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2 mercaptopropionylglycine in plasma and urine,” Clin Chem 44:825–832; and Kuhara, et al. 1999. “Pilot study of gas chromatographic-mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease,” J Chromatog B 731:141–147). Despite the available methods for determining urine cystine concentration, it has been reported that urine cystine concentration alone was not an adequate predictor for cystine supersaturation (Nakagawa, et al. 2000. “Clinical use of cystine supersaturation measurements,” J Urol 164:1481–1485; and Pak, C. Y. C. and Fuller, C. J. 1983. “Assessment of cystine solubility in urine and of heterogeneous nucleation,” J Urol 129:1066–1070).
Clinical evaluation and treatment for cystine stone formation in a given cystinuric patient should be monitored by the assessment of urine cystine supersaturation. When cystine concentration in the urine exceeds cystine solubility, the urine is said to be supersaturated with cystine, which can lead to crystallization of cystine and stone formation. To determine the cystine supersaturation point of a urine specimen, one must know not only the cystine concentration but also the solubility of cystine in the urine. Nomograms relating cystine solubility to pH have been widely used in estimating urine cystine saturation (See Dent, C. E. and Senior, B. 1955. “Studies on the treatment of cystinuria,” Br J Urol 27:317–332; and Marshall, R. W. and Robertson, W. G. 1976. “Nomograms for the estimation of the saturation of urine with calcium oxalate, calcium phosphate, magnesium ammonium phosphate, uric acid, sodium acid urate, ammonium acid urate, and cystine,” Clin Chim Acta 72:253–260). Pak and Fuller and Nagakawa et al reported that (1) factors such as electrolyte and macromolecule concentration in a given urine sample can affect cystine solubility, (2) the electrolyte and macromolecular content is variable, (3) the saturation of urine with respect to cystine can not be accurately estimated by comparing a measured urinary cystine concentration to a cystine solubility curve such as that proposed by Dent and Senior, and (4) cystine saturation must be derived from the direct measurement of cystine solubility of a patient's urine sample. An empirical method was proposed wherein the original cystine concentration in a patient sample was measured, solid cystine added to an aliquot of the urine and incubated for 2 days at 37° C. keeping a constant pH, remaining solid cystine filtered from the urine sample, cystine concentration in the filtrate measured, and the original cystine concentration divided by the cystine concentration in the filtrate to give a cystine concentration ratio, wherein a value of 1 represented saturation, a value of greater than 1 represented supersaturation, and a value of less than 1 represented undersaturation. In this assay, undersaturation was indicated by dissolution of the added cystine in the urine; and supersaturation was indicated by an increase in solid cystine (Pak, C. Y. C. and Fuller, C. J. 1983. J Urol 129:1066–1070; and Nakagawa, et al. 2000. J Urol 164:1481–1485).
While the empirical method described in these two papers provides an accurate measurement of urine cystine supersaturation, there is no consideration given to many of the factors which may affect the assay results. Crystallization of cystine during the collection of a 24-hour urine may lead to underestimation of cystine concentration and supersaturation (Kelly S. 1978. “Cystinuria genotypes predicted from excretion patterns,” Am J Med Genet 2:175–190). In addition, use of thiol-containing drugs such as D-penicillamine, tiopronin, and captopril complicate the measurement of urine cystine concentration and supersaturation (Lotz, M. and Potts, J. T. 1965. “Rapid, simple method for determining effectiveness of d-penicillamine therapy in cystinuria,” Br Med J 2:521; and Roesel, R. A. and Coryell, M. E. 1974. “Determination of cystine excretion by the nitroprusside method during drug therapy of cystinuria,” Clin Chim Acta 52:343–346). Colorimetric assays relying on a reduction reaction are greatly affected by these drugs. Even in HPLC and amino acid chromatography, techniques which are reported to allow assaying of both drug-bound and unbound cystine in treated patient, the conditions of these assay methods may alter cystine-drug binding so that the true amount of unbound cystine cannot be accurately determined. Thus, there is a need for methods to accurately measure urine cystine concentration and cystine supersaturation wherein the methods are not affected by collection and assay conditions. Further, the drugs used to treat cystinuria have a high incidence of serious adverse side effects, making it advantageous to prescribe the minimum effective dose for a given patient. Therefore, there is a need for methods that provide an accurate index of supersaturation while the drugs are in use.
A novel collection method and solid phase assay have now been found that provides an accurate measurement of urine cystine supersaturation, even when the patient is being treated with therapeutic cystine-binding drugs. The assay provides a means by which clinically reliable measures of overall treatment success in lowering the degree of urine cystine supersaturation and, therefore, risk of stone formation can be accurately assessed.